Islets of Langerhans are pancreatic micro-organs that play a central role in blood glucose homeostasis by secreting insulin, glucagon, and other hormones in a regulated manner. The overall goal of this research is to quantitatively determine the biochemical dynamics involved in glucose- stimulated insulin secretion from intact pancreatic islets. More specifically,, this study will address the role that cytoplasmic pH has on pyruvate transport into mitochondria, and will also address the role that cytoplasmic pH has on pyruvate transport into mitochondria, and will also address the role of mitochondria NADPH-shuttles in the insulin secretion process. In these studies, two-photon microscopy will be used to non-invasively monitor cellular NAD(P)H levels, with the ability to spatially separate signals from the cytoplasm and mitochondria. Metabolism will be correlated with beta-cell function by monitoring insulin secretion using established techniques such as radioimmuno assays and amperometry, or using fluorescence methods if feasible. Established fluorescence methodologies will be used to assay the cytoplasmic pH during nutrient stimulation. All studies will be mirrored in the NS-1 cell line, a classic cell culture model for beta-cells, which will allow a specific validations of this heavily used cell culture model.